QUICK SEARCH:   [advanced] Author: Keyword(s):
Home     Help     Feedback     Subscriptions     Archive     Search     Table of Contents

This Article Summary Full Text Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Englund, C. Articles by Samakovlis, C. Search for Related Content PubMed PubMed Citation Articles by Englund, C. Articles by Samakovlis, C.

Attractive and repulsive functions of Slit are mediated by different receptors in the Drosophila trachea

¹ Umeå Centre for Molecular Pathogenesis, Umeå University, S-901 87 Umeå, Sweden
² Department of Developmental Biology, Wenner-Gren Institute, Stockholm University, S-106 91 Stockholm, Sweden

View larger version (111K): [in a new window]   Fig. 1. Ablations of glial cells affect tracheal cell guidance. (A-C) Confocal projections showing the morphology of the ganglionic branch tip cell (GB1) visualized in green by the 1-eve-1 lacZ cytoplasmic marker. Robo staining, in red, was used to mark the longitudinal connectives. (A) At stage 15, GB1 has reached the ventral side of the neuropil and has extended several cytoplasmic processes. (B) At stage 16, GB1 turns at the CNS midline and extends one cytoplasmic protrusion dorsally and posteriorly along the dorsoventral channel glia to reach the dorsal side of teh neuropil. The GB1 nucleus has reached its position just ventral of the longitudinal connective. (C) By late stage 16, GB1 has extended laterally and posteriorly on the dorsal side of the neuropil. (D) Schematic drawing showing a ventral view of three pairs of GBs and their path in the wild-type embryonic stage 16 nerve cord. Inside the CNS, GB1 (dark blue) contacts first the ventral longitudinal glia (pale green). It turns before reaching the ventral midline and the midline glia (red), and migrates dorsally at the midline in close contact with channel glia (yellow). When GB1 reaches the dorsal side of the neuropil, it associates with the dorsal longitudinal glia (dark green) and extends laterally and posteriorly. The stalk cell of the ganglionic branch, GB2 is shown in pale blue, longitudinal axon tracts in brown and the borders of the CNS with broken lines. (E-I) GBs are misrouted when longitudinal glia are ablated by the expression of UAS-ricinA. The tracheal lumen of stage 16 embryos is shown in blue and the longitudinal and midline glia cells in E,F or the longitudinal connectives in G-I in brown. (E) Longitudinal and (F) midline glial cells are shown by the expression of UAS-lacZ driven by C321c-GAL4 and by Mz520-GAL4, respectively. (H) When longitudinal glia are ablated GBs stall outside CNS (arrow) or turn to migrate posteriorly before crossing the longitudinal tracts (arrowhead). Ablation of midline glial cells leads to midline crosses (I) of the GBs (arrows) and the longitudinal axon tracts (arrowhead) (compare with G). All panels show ventral views, anterior is towards the left. Scale bars: 4 µm in A-C; 8 µm in E-I.

View larger version (72K): [in a new window]   Fig. 2. Expression of Slit and its receptors during tracheal development. Confocal analysis of wild-type embryos carrying the UAS-EGFPF marker expressed under the control of the tracheal-specific driver btl-GAL4, double stained with anti-GFP to visualize the tracheal cells in green and anti-Slit (A,B), anti-Robo (C) or anti-Robo2 (D,E) in red. (A) Slit expression at the ventral midline close to the migrating tracheal tip cells of the ganglionic branch (top) and at the dorsal side, in cells just underneath the migrating dorsal branches (bottom, dorsal view). Dotted lines indicate the part of the tracheal dorsal branches that is out of focus. (B) Slit expression at the contacts of the gut epithelium and the tracheal visceral branches (anterior is upwards). (C-E) Lateral views showing the expression of Robo and Robo2 in the invaginating tracheal cells at stage 11 (C,D, top), the dorsal trunk and dorsal branch at stage 13 (C,D, bottom). (E) Robo2 expression in the growing visceral branches. (F) Summary drawings of Robo and Robo2 expression in different branches (DB, dorsal branch; DT, dorsal trunk; VB, visceral branch; GB, ganglionic branch) at stages 12 and 16. At stage 12 (top) both Robo (black) and Robo2 (gray) are expressed in cells of the DT, but Robo2 is also expressed in the DB and the VB. After stage 13, Robo is no longer detectable in the trachea, whereas Robo2 is expressed in DT and DB until the end of embryogenesis (bottom). Scale bars: 8 µm in A-E.

View larger version (102K): [in a new window]   Fig. 3. Slit signaling is required for ganglionic branch turning at the midline. (A-M) Stage 16 embryos stained for the tracheal lumen in blue and the GB1 cell marker DSRF (A-E) or longitudinal connectives (F-K) in brown. In wild-type embryos (A), GBs turn posteriorly at the ventral midline but in robo and slit mutants (arrows in B and D) they cross it. The fascicles of the longitudinal connectives are separated in wild type (F), but cross the midline in robo mutants (arrows in G); in slit mutants (I), the fascicles are fused at the midline (arrows). (L) Transgenic tracheal expression of UAS-robo (anti-Robo staining in brown) in robo mutants rescues GBs from crossing the ventral midline (arrowhead), whereas GBs with no detectable Robo expression still cross (arrow). The axon phenotype in robo mutant embryos is partially rescued by transgenic expression of UAS-robo in all neurons with the elav-GAL4 driver (K), but GBs still cross the ventral midline (arrow in K). (C) In robo2 mutant embryos, the GBs fail to enter the CNS (asterisk) and several of the GBs that do enter stall or become misrouted (arrowhead). (H) The longitudinal axon tracts are disrupted (arrows) and the outer fascicles fuse with the medial fascicles in robo2 mutants. (M) The GB failure to enter the CNS in robo2 mutants can be rescued by transgenic tracheal expression of UAS-robo2 with the SRF-GAL4 driver. (E) GBs in the double mutant robo, robo2 fail to enter the CNS (asterisk) and cross the ventral midline (arrow) and the longitudinal axons collapse along the midline (J). These phenotypes are very similar to the phenotypes of slit mutants (D,I). Scale bar: 20 µm in A-M.

View larger version (93K): [in a new window]   Fig. 4. Overactivation of the Slit signaling pathway in the CNS can repel the ganglionic branches. (A-H) Stage 16 embryos stained to visualize the tracheal lumen in blue and the longitudinal axons (in A,B,E,F) in brown. (B) In comm mutants, GBs turn to migrate posteriorly before they reach the midline but after crossing the longitudinal tracts. The longitudinal axon tracts are further apart presumably due to the absence of commissures. (C) Ectopic expression of Robo in the GB1 cell (Robo staining in brown) using the SRF-GAL4 driver results in GBs turning posteriorly before they cross the longitudinal tracts (arrow). GBs that do not overexpress Robo migrate closer to the midline before turning posteriorly (arrowhead). (D) Early turning of GB1 before the longitudinal axons (arrow) can also be seen in embryos with ectopic expression of robo2 (embryos stained for a HA-tagged Robo2 in brown). Several of the Robo2 overexpressing GBs still reach the midline (arrowhead) in contrast to all the GBs that turn prematurely when they overexpress Robo in C. (E) Ectopic expression of Slit in the longitudinal glia using the C321c-GAL4 driver leads to premature turning (arrow) and stalling (arrowheads) of GBs. (F) Several GBs cross the ventral midline (arrows) in response to general tracheal misexpression of comm using the btl-GAL4 driver. (G) UAS-robo2 overexpression in GB1 using the SRF-GAL4 driver cannot rescue the GB midline crossing phenotype in robo mutants (arrows, embryos stained for HA-tagged Robo2 and the tracheal lumen). UAS-robo overexpression with the same driver can rescue the GB CNS entry phenotype in robo2 mutants (H; embryos stained for Robo-HA and tracheal lumen). Scale bar: 20 µm in A-H.

View larger version (121K): [in a new window]   Fig. 5. Slit signaling is required for the outgrowth of tracheal branches outside the CNS. Lateral view of stage 16 embryos carrying one copy of the 1-eve-1 lacZ marker stained to visualize tracheal cells and lumen. In robo2 (B) and slit (C) mutant embryos outgrowth of the dorsal branches (DBs) is disrupted, resulting in missing (asterisks) or short DBs. In robo mutants (A), DB outgrowth is not affected. Several of the GBs in robo2 (E) and slit (F) mutants are shorter and misrouted (asterisks) In robo mutants (D), GBs migrate ventrally as in wild-type embryos (arrow in D). Scale bar: 20 µm in A-F.

View larger version (123K): [in a new window]   Fig. 6. Tracheal branches are attracted to sites of ectopic Slit expression. (A-J) Embryos stained to visualize the tracheal lumen and double stained for Slit (A,B) and ß-galactosidase (D,E), to visualize the sites of ectopic Slit and ß-gal expression. (A) Ectopic expression of Slit in epidermal stripes, induced using the en-GAL4 driver, disrupts the anterior and posterior elongation of the dorsal trunk (DT) branches (arrows in B); the dorsal trunk appears to extend dorsoventrally along the en-GAL4 stripes. (C) Removing endogenous Slit expression results in a stronger DT phenotype. (D) ß-Gal expression driven by twi-GAL4 shows that the driver directs UAS-transgene expression in the gut epithelia at stage 13, when the tracheal visceral branches (VBs) (E) start migrating towards and along the surface of the gut. The broken line indicates the terminal cells of the VB that are in close contact with the gut. (H) In wild-type embryos, the VBs from metamere T2 and T4 migrate towards the anterior part of the midgut. The T3 metamere does not form a VB. (I) Ectopic expression of UAS-slit with the twi-GAL4 driver can induce formation of additional VBs from metamere T3 (arrow in I). In robo mutants, the ectopic expression of Slit results in formation of additional VBs from T3 and also from other metameres (arrows in J,G; dorsal and lateral views, respectively). The increased level of branch formation in response to ectopic Slit expression cannot be detected in robo2 mutants (F). Scale bars: 20 µm.